Two-photon excitation microscopy of tryptophan- containing proteins M. Lippitz†, W. Erker‡, H. Decker‡, K. E. van Holde‡§, and T. Basche´ †¶ †Institute of Physical Chemistry, Jakob-Welder-Weg 11, and ‡Institute of Molecular Biophysics, Jakob-Welder-Weg 26, University of Mainz, 55099 Mainz, Germany Contributed by K. E. van Holde, December 11, 2001 We have examined the feasibility of observing single protein oxygen–copper complex gives rise to two ligand-to-metal molecules by means of their intrinsic tryptophan emission after charge-transfer absorption bands that are centered at 340 nm (␧ two-photon excitation. A respiratory protein from spiders, the Ϸ 20,000 MϪ1⅐cmϪ1 per binding site; ref. 27) and 570 nm (␧ Ϸ 24-meric hemocyanin, containing 148 tryptophans, was studied in 1,000 MϪ1⅐cmϪ1). The short-wavelength absorption band over- its native state under almost in vivo conditions. In this specific case, laps with the Trp emission band and results in an almost the intensity of the tryptophan emission signals the oxygen load, complete quenching of the Trp fluorescence because of Fo¨rster allowing one to investigate molecular cooperativity. As a system transfer (28, 29). Therefore, in Hc the intensity of the Trp with even higher tryptophan content, we also investigated latex fluorescence monitors the oxygen load (25) and can therefore be spheres covered with the protein avidin, resulting in 340 trypto- used for obtaining oxygen binding curves (23, 25) and to clarify phans per sphere. The ratio of the fluorescence quantum efficiency the mechanism of the cooperative oxygen binding. Considering to the bleaching efficiency was found to vary between 2 and 180 the above features, Euryplema Hc was identified as an appro- after two-photon excitation for tryptophan free in buffer solution, priate candidate for our investigations. First, it contains 148 Trp in hemocyanin, and in avidin-coated spheres. In the case of hemo- residues (22), and second, the Trp emission intensity monitors cyanin, this ratio leads to about four photons detected before the oxygen load. Therefore, experiments at the single protein photobleaching. Although this number is quite small, the diffusion level promised—besides the envisioned imaging—to allow the of individual protein molecules could be detected by fluorescence study of conformational transitions assumed to occur between correlation spectroscopy. In avidin-coated spheres, the trypto- different levels of oxygenation of Hc. phans exhibit a higher photostability, so that even imaging of To overcome the problem of direct UV excitation of Trp, we single spheres becomes possible. As an unexpected result of the have used two-photon excitation (TPE) (30, 31), which was measurements, it was discovered that the population of the previously applied to single-molecule detection by Mertz et al. oxygenated state of hemocyanin can be changed by means of a (32). This technique promises to selectively excite transitions in one-photon process with the same laser source that monitors this the UV by visible pulsed laser sources without the broad population in a two-photon process. unspecific background of conventional, one-photon excitation (OPE) in the UV. Compared with fluorescent dyes (33) that y eliminating ensemble averaging, single-molecule fluores- were studied in the first two-photon experiments (32), Trp has Bcence microscopy and spectroscopy (1–4) have proven to a rather low two-photon absorption cross section (34). This obtain novel insights into the dynamics of complex heteroge- disadvantage seems to be partly compensated by the large neous systems (5–7). In the field of biological applications, number of Trps in a single Hc. Another important issue is the confocal scanning and wide-field fluorescence microscopy of photostability of Trp after TPE. To get an idea about the single protein molecules have been used to study conformational photobleaching quantum efficiency of Trp in different environ- transitions (8–10), enzyme kinetics (11), local pH in cells (12), ments besides Hc, we also studied unbound Trp and the Trp- and diffusion in membranes (13) and cells (14) (for a review see containing protein avidin bound to latex spheres. ref. 15). Although the most general single molecule application Materials and Methods relies on the use of fluorescent dyes, which are attached to the biomolecule of interest, the intrinsic fluorescence emission of Hc of the tarantula E. californicum was purified and tested ⅐ biomolecules has also been utilized in some cases (16–18). according to ref. 23. The buffer used was 0.1 M Tris HCl at pH In the present study, we have explored the potential of 7.8 in the presence of 5 mM CaCl2 and 5 mM MgCl2. Trp fluorescence detection of single proteins by making use of the purchased from Fluka (Neu-Ulm, Germany) was dissolved in the intrinsic tryptophan (Trp) emission. Although aromatic amino same buffer and used without further purification. For control acids as Trp or tyrosine are abundant in many biopolymers, to experiments, Hc was labeled with tetramethylrhodamine (TMR; ␮ our knowledge there have been no reports on single-molecule Molecular Probes). Ten parts Hc solution (2 M) in borate ͞ ͞ ͞ detection of Trp or proteins containing Trp. Actually, there are buffer (25 mM borate NaOH, pH 8.5 5 mM CaCl2 5mM a number of reasons for the lack of reports, mainly relating to the MgCl2) was mixed with three parts of dye solution (1 mM) and ␭ ϭ problem of excitation in the UV ( max 280 nm) and the limited incubated for 2 h in the dark. The Hc was purified by gel filtration photostability of UV-absorbing chromophores like Trp. and ultracentrifugation in Tris buffer. Latex spheres (diameter: Because of the supposedly low photostability of Trp, we have 40 nm) crosslinked on their surface with the protein avidin concentrated on systems that contain a large number of Trps. (Fluospheres, Molecular Probes) were studied as another model One particular class of material we have investigated are hemo- cyanins, which are large multisubunit proteins functioning as Abbreviations: Hc, hemocyanin; TMR, tetramethylrhodamine; OPE, one-photon excitation; respiratory proteins in the hemolymph of arthropods and mol- TPE, two-photon excitation; FCS, fluorescence correlation spectroscopy. lusks (19–21). One of the hemocyanins best investigated is the ϫ §Permanent address: Department of Biochemistry and Biophysics, Oregon State University, 4 6-meric hemocyanin (Hc) from the tarantula Eurypelma Corvallis, OR 97331. californicum. The complete sequences of the subunits for this ¶To whom reprint requests should be addressed. E-mail: [email protected]. species are known (22), and the oxygen binding is well under- The publication costs of this article were defrayed in part by page charge payment. This stood (23–25). One molecule of oxygen is reversibly bound article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. between two copper atoms in a transverse coordination (26). The §1734 solely to indicate this fact. 2772–2777 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052662999 Downloaded by guest on September 30, 2021 Fig. 1. Diagram of the experimental setup for two-photon microscopy. The laser system consists of an argon-ion-laser (Arϩ), a titanium-sapphire laser (Ti:Sa), and an optical parametrical oscillator (OPO). The light beam is atten- uated (Attn), filtered (F1), and expanded (BE) before it is focused by a microscope objective (Obj) onto the sample. The fluorescence light is collected by the same objective, reflected by a dichroic beamsplitter (DC), filtered (F2), and detected by a photomulitplier tube (PMT) or a spectrometer and charge- coupled device camera (CCD). system. Avidin is a small homotetrameric protein with a molar mass of 66 kg/mol. Each subunit has the ability to bind one molecule of biotin and contains four Trp residues (35). From the biotin-binding capacity given by the manufacturer, the number of Trps per sphere is calculated to be 340. The same avidin- coated spheres with a dye filling have been used for control experiments. Standard emission spectra were measured in a commercial spectrometer (F-4500, Hitachi, Tokyo) at an excitation wave- length of 295 nm. For TPE at 590 nm (34), Fourier-limited pulses of 180 fs were produced by an optical parametrical oscillator (OPO) with intracavity frequency doubling (Angewandte Physik und Elektronik, Berlin), which was pumped by an argon-ion͞ Fig. 2. Excitation intensity dependence of the Trp fluorescence count rate. titanium-sapphire laser source (Coherent Radiation, Palo Alto, (A)A1mMTrpsolution (buffer) shifted by a factor of 5 for clarity, a 50 nM ␮ CA; see Fig. 1). The light was focused by a microscope objective solution of avidin-coated spheres (avidin), and 2 M Hc solutions in the ϫ ϭ oxygenated (Hcoxy) and deoxygenated state (Hcdeoxy). Background fluores- (Ultrafluar 100 , numeric aperture 1.2, Zeiss) onto the cence is subtracted. (B and C) Ratios of the measured to the expected fluo- sample. The fluorescence emission was collected by the same rescence count rate. The data are fitted with models discussed in the text. objective and separated from the excitation light with a custom- made dichroic beam splitter and filter set with a transmission window from 310 to 380 nm (both AHF Analysentechnik, in Hc and compared it to Trp in buffer solution and in avidin- Tu¨bingen, Germany). The fluorescence light was either detected coated spheres. Fig. 2A shows the intensity dependence of the by a Peltier-cooled photomultiplier (R4220P, Hamamatsu, Mid- fluorescence count rate after TPE for four different samples: a dlesex, NJ) or analyzed with a monochromator and a liquid 1 mM Trp solution, a 50 nM solution of avidin-coated spheres ␮ N2-cooled charge-coupled device camera (Roper Scientific, and two 2 M solutions of Hc in the oxygenated (Hcoxy) and the Trenton, NJ).